Anaerobe 20 (2013) 27e31

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Anaerobe
journal homepage: www.elsevier.com/locate/anaerobe

Clinical microbiology

Study of the effects of chitosan upon Streptococcus mutans adherence and biolm
formation
E.M. Costa, S. Silva, F.K. Tavaria, M.M. Pintado*
CBQF - Centro de Biotecnologia e Qumica Fina Laboratrio Associado, Escola Superior de Biotecnologia, Universidade Catlica Portuguesa/Porto, Rua Dr. Antnio Bernardino
Almeida, 4200-072 Porto, Portugal

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 25 September 2012
Received in revised form
14 February 2013
Accepted 19 February 2013
Available online 27 February 2013

The main aim of this work was to access the potential use of high and low molecular weight chitosans as
potential oral antimicrobials, particularly as antibiolm agents. Chitosans interference with Streptococcus
mutans capability to adhere and form biolms was assessed. Additionally the effect upon mature and
polymicrobial biolms was also evaluated. The results obtained showed that chitosan was capable of
interfering with S. mutans adhesion and primary biolm formation. This action was observed up to a
week with little to none decrease in efciency. In addition chitosan was capable of inhibiting biolms
formed by two microorganisms and was capable of acting upon mature biolms leading to signicant
reductions (94%) in biolm survival. However clear statistical differences (p < 0.05) were registered in all
assays with, in most assays, HMw chitosan presenting higher efciency than LMw chitosan. Considering
this results chitosans potential as a valid alternative to traditional antimicrobials in oral health its
evident.
2013 Elsevier Ltd. All rights reserved.

Keywords:
Streptococcus mutans
Adherence
Biolm
Mature biolm
Chitosan
Sub-MIC

1. Introduction
Oral diseases associated to dental biolms affect the majority of
the worlds population with Streptococcus mutans being a key
contributor to the development of those diseases due to its role on
biolm formation [1]. S. mutans, despite not being a primary
colonizer, has a recognized role in the initiation of dental caries
[2,3] being a key contributor to the formation of the biolm associated with dental caries [1,4e6]. Furthermore the ability of
S. mutans to utilize dietary sucrose to produce exopolysaccharides
(EPS), which act as anchoring points for other bacteria (secondary
colonizers) like Lactobacillus acidophilus and Aggregatibacter actinomycetemcomitans to adhere, cements. S. mutans as one of the
main contributors to the development of oral diseases [1,4e8].
Until now, several substances have been tested for the control of
oral biolm including essential oils, amine uoride, triclosan, etc.
but the most thoroughly tested and more effective known antibiolm agent is chlorhexidine [9]. However, chlorhexidine has been
associated to some secondary effects, namely the reduction of human taste perception and the pigmentation of oral tissues, which
limits its application [9e12]. Therefore, the search for new

* Corresponding author. Tel.: 351 225580097; fax: 351 225090351.

E-mail address: mpintado@porto.ucp.pt (M.M. Pintado).
1075-9964/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.anaerobe.2013.02.002

antimicrobials has arisen, and natural products are favorite due to

the lack of secondary effects and possible long term usage in the
oral cavity.
Chitosan, a natural polysaccharide derived from chitin, has a
proven antimicrobial activity against a wide variety of microorganisms including fungi, algae and bacteria which is affected by a
number of factors including chitosans molecular weight and degree of deacetylation [13,14]. Moreover chitosan possesses bioadhesive capabilities thus making it capable of residing in the oral
cavity [15]. Most of the published works concerning the effects of
chitosan report its bactericidal action against planktonic microorganisms, but limited information is known about its activity upon
adhesion and biolm formation [9,16,17].
The aim of our work was to assess the impact of sub-MIC concentrations of high and low molecular weight chitosan upon
S. mutans adhesion, biolm formation and mature biolms.

2. Material and methods

2.1. Microorganisms
Streptococcus mutans (CCUG 45091), L. acidophilus (CCUG 33386)
and A. actinomycetemcomitans (CCUG 13227) were obtained from
the culture collection of the Gteborg University (CCUG) (Sweden).

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E.M. Costa et al. / Anaerobe 20 (2013) 27e31

All microorganisms were grown in Wilkins-Chalgrens broth (Oxoid,

England) and all inoculums were prepared by inoculating two
colonies of each strain in 10 ml of WC broth and grown overnight at
37  C.

described by Miles [20], in Wilkins-Chalgrens agar (Oxoid, Hampshire, England). Plates were then incubated at 37  C for 24 h under
anaerobic conditions. Results were given as inhibition percentages
using the following formula:

2.2. Sources and preparation of chitosan solutions

%I 100  Log CFU sample=Log CFU control  100

High and low molecular weight chitosan were obtained from

SigmaeAldrich (St. Louis, USA). High molecular weight chitosan
was characterized by a DD >75% and a Mw of 624 kDa. Low molecular weight chitosan was characterized by a DD between 75 and
85% and a Mw of 107 kDa.
Since these chitosans are only soluble at low pH, chitosan solutions were prepared in a 1% (v/v) solution of glacial acetic acid
99% (Panreac, Barcelona, Spain) corresponding to a nal pH of 3.
Chitosan was added to 1% acetic acid to the desired concentration.
Afterwards, the solution was stirred overnight at 50  C to promote
complete dissolution of chitosan. The pH was adjusted with NaOH
(Merck, Darmstad, Germany) to a nal value of 5.6e5.8 and solutions were stored at refrigerated temperature. Chitosan sub-MIC
concentrations in every assay were obtained via dilution of chitosan solutions in sterile full strength WCB with 5% (w/v) sucrose.
2.3. Tube test
The effect of chitosan on biolm formation was screened by
adapting the procedure described by Christensen [18]. Transparent
25 ml plastic tubes containing chitosan (HMw and LMw) and WCB
with 5% sucrose to a nal volume of 5 ml were inoculated at 1% (v/
v). Chitosan was added at the sub-MIC concentrations of 2 and
1 mg/ml for HMw and of 4 and 2 mg/ml for LMW selected
accordingly to the MIC results reported by Costa [19]. Each tube was
incubated in horizontal position at 37  C for 72 h under anaerobic
atmosphere. Tubes without chitosan and inoculated under similar
conditions and non-inoculated medium with chitosan were used as
positive and negative controls, respectively.
After incubation, the contents of each tube were carefully
removed and discarded. Subsequently, 2 ml of crystal violet solution (Merck, Darmstad, Germany) were added to each tube and left
to react for 5 min on a roll and tilt mixer (Movil-Rod, J.P. Selecta,
Barcelona, Spain) for homogenous staining. After that, the staining
solution was discarded and the tubes inverted and left to dry,
overnight, at room temperature.
Qualitative evaluation of biolm formation was assessed by
observation of a visible lm lining the walls of the tube, and the
amount produced was tentatively estimated as absent (score 0),
weak (score 1), moderate (score 2) or strong (score 3). All assays
were done in triplicate.
2.4. Adherence
The effect of chitosan on bacterial adhesion to surfaces was
tested by using 24 wells microplates. Briey, 1 cm aluminum disks
were dipped for 30 or 90 s in a well containing either 1% (v/v) HMw
or LMw chitosan. Chitosan was used at full force, and not sub-MIC
concentrations, in order to assure the best possible coating of the
aluminum disks. Following that disks were rinsed with sterile
water and submerged in a well containing inoculum for 60 s, after
which they were placed in wells containing the appropriated medium and incubated for 24 h at 37  C. Two controls were simultaneously assessed. In the rst, disks were dipped in sterile water and
then inoculated and incubated. In the second, disks were dipped in
the test solutions and after rising in sterile water they were incubated without inoculum. After 24 h disks were recovered and after
serial dilutions viable counts were assessed by drop method, as

All assays were performed in duplicate.

2.5. Microtiter-plate test
Quantication of biolm production in batch and fed-batch was
carried out by adapting the protocol of Ref. [21]. Briey, in a at
bottom 96 microplate wells (Nunc, Roskilde, Denmark) were lled
with 200 ml of test solutions with chitosan added at sub-MIC concentrations. Biolms were formed in a batch and in a fed-batch
mode. In the rst the plate was incubated at 37  C for 48 h in a
rectangular jar under anaerobic conditions. In the second the medium was renewed every 24 h. This assay had the duration of 96
and 198 h at 37  C incubated under anaerobiosis.
To visualize biolms, the contents of each well were discarded
and then washed 3 times with sterile deionized water in order to
remove non-adherent cells. The remaining attached bacteria
were xed with 200 ml of ethanol (Panreac, Barcelona, Spain) for
15 min, ethanol was then discarded and the wells were air dried.
After that, 200 ml of crystal violet solution (Merck, Germany)
were added to the wells for 5 min. Excess stain was removed by
rinsing the plate under tap water followed by air drying of the
plate.
Adherence was quantied by measuring the OD at 630 nm using
a microplate reader.
All experiments were done in triplicate for each bacterium. OD
values from wells containing un-inoculated WCB were used as
negative controls. A positive control where chitosan solution was
replaced by sterile deionized water for each bacterium was used.
Results for this test were given as a percentage of biolm formation inhibition applying the following formula:

% biofilm formation inhibition

100  ODassay =ODcontrol  100

2.6. Dual-species biolms

Quantication of the effect of chitosan upon biolms formed by
two different bacteria (S. mutans and L. acidophilus; S. mutans and
A. actinomycetemcomitans) was evaluated using the biolm
microtiter-plate assay as described above with one modication.
The test solutions were inoculated with 1% (v/v) of each
tested bacteria to achieve a 2% (v/v) inoculum concentration. Results were obtained as referred above and all assays were done in
triplicate.
2.7. Mature biolms assay
The assessment of the effect of chitosan upon mature biolms
was performed by adaptation of the protocol of Ref. [21]. Briey,
in a at bottom 96 microplate wells were lled with 200 ml of
medium with inoculum at 1% and then incubated for 48 h at
37  C in anaerobiosis to assure full maturation of the biolm.
After 48 h the medium was carefully aspirated and the wells
were rinsed with phosphate buffer. Following that 200 ml of
medium with chitosan at sub-MIC concentrations were added to
the wells and incubated at 37  C for 48 h under anaerobic
conditions.

E.M. Costa et al. / Anaerobe 20 (2013) 27e31

Biolm visualization and quantication was performed as

described above for the microtiter-plate test.
2.8. Statistical treatment
The statistical differences observed for each type of chitosan and
microorganisms for each method were evaluated using IBM SPSS
Statistics v.19.0.0 (New York, USA). The normality of the results
distribution was evaluated through ShapiroeWilks test. The differences were evaluated using an One way ANOVA test associated
with Scheffe test (for normal distributions) or the non-parametric
ManneWhitney test for the rest. The differences were considered
signicant with p  0.05.
3. Results
3.1. Tube test
The results obtained from the test tube allowed us to screen the
potential of chitosan as an inhibitor of S. mutans adherence and
biolm formation. In all assayed concentrations both chitosans
molecular weights were capable of completely inhibiting biolm
formation (data not shown) at sub-MIC concentrations with no
differences in activity being found between and of the MIC.
3.2. Adherence
Adherence is the rst and crucial step in biolm formation in the
oral cavity as it permits the establishment of microorganisms in the
oral cavity [2]. Exposure of aluminum disks to chitosan for 30 or
90 s (Fig. 1) thus simulating the lowest and the highest rinsing time
normally used in mouthwashes, presented high inhibition percentages of S. mutans adhesion, with inhibition percentages
reaching 100% for HMw chitosan and ranging between 94 and 99%
for LMw chitosan. Statistical analysis of the results shows that for
HMw there are no signicant differences between 30 and 90 s
exposure time in terms of adhesion inhibition with both conditions
presenting equally high inhibition percentages. On the other hand
for LMw chitosan there were clear differences between 30 and 90 s
of contact time, with the latter presenting signicantly higher inhibition percentages. Additionally, there were clear differences
(p < 0.05) between Mw for 30 s contact time with HMw chitosan
showing signicantly higher inhibition percentages. Considering

Fig. 1. Schematic representation of the adherence method.

29

that S. mutans has a recognized adherence capability, due to its

glucosyltransferases and fructosyltransferases, and a capability to
rapidly synthesize exopolysaccharides [1,2], our results show that
chitosan has the potential to minimize S. mutans colonization in the
oral cavity. Furthermore, these results are in line with those obtained by Tarsi, Muzzarelli [2] who showed that sub-MIC concentrations of chitosan were capable of diminishing S. mutans adhesion
to hydroxyapatite up to 66%.
3.3. Microtiter-plate test
In this quantitative assay the purpose was to assess the impact
of chitosan upon biolm formation and evaluate activity up to a
weeks time. The results obtained (Fig. 2) show that chitosan was
capable of inhibiting biolm formation up to one week (168 h) with
inhibition percentages reaching up to 91%. Higher concentrations of
both Mws presented statistically superior (p < 0.05) results at 48 h
implying that chitosan concentration is a key factor in opposing
initial adhesion and consequent biolm formation. Furthermore
HMw chitosan presented both at and of the MIC signicantly
higher (p < 0.05) activity than LMw chitosan. At 96 h results show
that LMw chitosan presented slightly higher inhibition than HMw
chitosan. At this time the only statistically different (p < 0.05) result
was obtained for 1 mg/ml HMw, which presents a signicant lower
inhibition percentage. Finally, at 168 h the results obtained show
that, at this time, there are no signicant differences, either between Mw and concentrations, in terms of S. mutans biolm formation, with both Mw presenting very similar inhibition
percentages (ca. 90%). It is interesting to note that in all the assays
the only signicant differences found between Mw were for the
48 h assay, with HMw presenting signicantly higher inhibition
percentages. These results imply that Mw may not be a signicant
factor in chitosans antibiolm activity against S. mutans and that
may be due to the presence of the EPS produced by S. mutans, which
will constrain the interaction between the larger molecules of
HMw chitosan and the bacteria. Our results are in line with those
reported by Bae et al. [9]and Busscher et al. [22] who showed that
chitosan reduced biolm viability and reduced plaque index.
However, these authors only tested LMw chitosans against
S. mutans biolm formation and Bae et al. [9] chitosans was
chemically altered making it water soluble. As such, despite being
similar, previous results are limited in scope as they only present a
limited view of chitosans action. Furthermore this method presents
some limitations as the manipulation process involved in the
staining of the biolm may lead to higher or lower inhibition results
as a vigorous cleaning step may lead to destruction of the biolm in
the wells. On the other hand a not strong enough cleaning step may

Fig. 2. Effect of sub-MIC concentrations of chitosan upon biolm formation

throughout time. (Batch assay e 48 h; Fed-batch assay e 96 and 168 h). Different
letters represent the statistical differences found.

30

E.M. Costa et al. / Anaerobe 20 (2013) 27e31

lead to the persistence of loosely adhered cells that will alter the
nal results. Furthermore the need to kill the biolm (xation with
alcohol in order to stain it) will lead to an impossibility to discern
between alive and dead cells present in said biolm.
3.4. Dual-species biolms
In an effort to gain a better insight into chitosans potential to
prevent the formation of multi-species biolms we studied the
effect upon the interaction of S. mutans with two known secondary
colonizers, L. acidophilus and A. actinomycetemcomitans. Since
S. mutans is one of the main biolm producers we used S. mutans
sub-MIC.
As can be seen in Fig. 3 there is a clear decrease in chitosan
biolm inhibition percentages in comparison to the results obtained for the single species biolms of S. mutans (Fig. 2). Low
molecular weight chitosan showed clear differences (p < 0.05) in
behavior between the two different polymicrobial biolms in study
presenting higher inhibition percentages when in contact with
S. mutans and A. actinomycetemcomitans than when in the presence
of S. mutans and L. acidophilus. Considering that LMw chitosan acts
better upon Gram-negative bacteria and vice versa [13], it is only
natural that the polymicrobial biolm constituted by two Grampositive bacteria is less affected by LMw chitosan action.
Regarding HMw chitosan the inhibition percentages obtained
were superior to 50% in all assayed conditions for both polymicrobial
biolms in study. The only statistical difference was found for the
of the MIC where the S. mutans and L. acidophilus biolm presented
higher percentages of biolm formation inhibition. It is interesting to
note that despite expecting HMw chitosan to present higher activity
against the biolm constituted only by Gram-positive bacteria, this
did not occur. In fact, for the of the MIC there were no statistically
signicant differences between both polymicrobial biolms. These
results lead to the conclusion that in the case of HMw chitosan the
concentration will be a signicant factor in chitosan activity
depending on whether the affected polymicrobial biolms are
constituted only by Gram-positive bacteria or by Gram-positive and
Gram-negative bacteria. Overall there are clear differences (p < 0.05)
between Mws when in presence of polymicrobial biolms constituted only by Gram-positive bacteria with LMw chitosan presenting
signicantly lower results than HMw chitosan. Despite various
studies regarding the importance of S. mutans on secondary

Fig. 4. Effect of sub-MIC concentrations of chitosan upon S. mutans mature biolms.

Different letters represent the statistical differences found.

colonizers biolm formation [23,24]there are no previous studies

showing the impact of chitosan upon polymicrobial biolms. Our
results show that despite presenting a clear loss in inhibition capability, chitosan is nevertheless still capable of inhibiting, in average,
60% of biolm formation.
3.5. Mature biolms assay
Reduction or elimination of already formed biolms is a difcult
challenge for all antimicrobials, since biolm structures represent
an anchor structure and protection for microorganisms. Previously
Orgaz et al. [25] showed that 1 h exposure of 1% (w/v) medium
molecular weight (MMw) chitosan was capable of causing a 6 log
viable cell reduction in Listeria monocytogenes and a 3e5 log to
other microorganisms (Pseudomonas uorescens, Salmonella enterica and Bacillus cereus). In our assay we exposed S. mutans mature
biolms to sub-MIC concentrations of LMw and HMw chitosan for
48 h and as can be seen in Fig. 4there are clear differences (p < 0.05)
in behavior between Mw and concentrations. Low molecular weight
chitosan showed inhibition percentages inferior to 30% for both
tested concentrations, while on the other hand HMw chitosan inhibition percentages were ca. 95 and 88% for both concentrations
tested. Considering chitosans cationic nature and, as previously
referred, the inuence of the Mw in chitosans activity we can
conclude that the great difference in values between HMw and
LMw was only due to the fact of S. mutans being a Gram-positive
bacteria and that chitosans Mw will be the key factor in its action
when acting upon mature microbial biolms. This behavior may be
due either to chitosans high positive charge being capable of
interacting and interfering with the biolm structure or to its DD.
However in the present study this variable was not assessed with
both chitosans used possessing similar DD values.
4. Conclusions

Fig. 3. Effect of S. mutans sub-MIC concentrations of chitosan upon dual-species biolm formation. e S. mutans and L. acidophilus biolm formation; e S. mutans and
A. actinomycetemcomitans biolm formation. Different letters represent the statistical
differences found.

Chitosan showed an evident strong effect against S. mutans

adherence and biolm formation demonstrating its action through
three different steps inhibiting initial adherence and biolm formation and disrupting mature biolms. Furthermore chitosan was
capable of inhibit dual-species biolm formation, with higher efcacy for HMw, thus indicating that chitosan may be able to act
upon more complex, multi-species, biolms. Lastly chitosan
showed activity upon mature biolms, with HMw presenting
higher activity, thus revealing that chitosan could possibly be used

E.M. Costa et al. / Anaerobe 20 (2013) 27e31

as a means to reduce dental plaque and the consequent pathologies. However caution is required when analyzing this results as
the inuence of the acquired pellicle, in particular of mucins to
which S. mutans is known to adhere [5], upon the chitosan action
was not evaluated. As such further studies are still required to
better understand chitosans action. Moreover all the experiments
were performed under static conditions and as such the inuence
of the salivary ow was also not assessed. As such further studies
will be required.
Nevertheless chitosan demonstrates great promise as a possible,
natural, plaque control agent to be included in new formulations
for oral applications, not only as antimicrobial agent but also for
biolm control.
Acknowledgments
The author hereby gratefully acknowledges the Agency of
Innovation (Agncia de Inovao, ADI, Portugal) and Quadro de
Referncia Estratgico Nacional (QREN, Portugal) which through
the project QUITORAL e Desenvolvimento de novas formulaes
de quitosanos com aplicao em medicina oral (QREN-ADI 3474)
and the National Funds from FCT e Fundao para a Cincia e a
Tecnologia through project PEst-OE/EQB/LA0016/2011provided
funding for the realization of this work.

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